Evaluating and predicting clinical outcomes by gene expression analysis

ABSTRACT

Methods of determining clinical outcomes in patients suffering from a pathological condition or syndrome are provided. Levels of intracellular gene expression re measured from a clinical sample provided by the patient, and the levels are compared to reference levels. Deviations from reference levels are predictive of clinical outcomes, for example, disease progression or response to therapeutic intervention.

FIELD OF THE INVENTION

[0001] The present invention relates to methods for evaluating andpredicting clinical outcomes in patients by measuring levels of geneexpression. Methods are provided for quantitating gene expressionlevels, and the measured levels are compared against reference levels.Deviations from the reference levels can be correlated with clinicaloutcomes. For example, the type and extent of a patient's response to atherapeutic intervention can be determined, or the prognosis for apatient's survival can be estimated. The gene expression levels can bemeasured in essentially any chosen body tissue or fluid. Surprisingly,it has been found that measurement of intracellular gene expressionlevels in blood are indicative of clinical outcomes.

BACKGROUND OF THE INVENTION

[0002] Methods for examining overall gene expression in, for example,disease states, previously have been described. See, for example, U.S.Pat. No. 5,874,219; Zakut et al., Cancer Research, 53:5-8 (1993);Mohaupt et al., Kidney International, 46:653-665 (1994); Liang andPardee (1992) Science 257, 967-971; Liang et al (1993), Nucleic AcidsRes. 21, 3269-3275; Bauer et al (1993) Nucleic Acids Res. 21, 4272-4280;Stone and Wharton (1994), Nucleic acid Res. 22, 2612-2618 and Wang andFeuerstein (1995) Biotechniques 18, 448-452; WO 93/18176 and DE 43 17414. These methods, however, generally provide a “snapshot” of geneexpression that is qualitative, rather than quantitative. Accordingly,the methods provide an indication only of whether a gene is beingexpressed at a detectable level in a particular tissue. Moreover, evenwhen applied to samples from patients suffering from a pathologicalsyndrome, none of these methods provides any correlation with clinicaloutcome for the patient. It is apparent, therefore, that new andimproved methods for measuring levels of gene expression and correlatingthose levels with clinical outcome are greatly to be desired.

SUMMARY OF THE INVENTION

[0003] There exists a need to determine and predict clinical outcomes inpatients It is therefore an object of the invention to provide methodsfor evaluating (e.g., determining and/or predicting) clinical outcomefor a patient suffering from a clinical condition or syndrome,comprising the steps of (a) providing a clinical specimen obtained orderived from the patient, (b) measuring the levels of expression of apreselected set of genes in the clinical specimen; and (c) comparingsaid levels of expression against a set of reference expression levels,where a deviation of the level of expression of one or more of thepreselected set of genes is indicative of clinical outcome for thepatient. The phrase “preselected gene(s)” refers to genes that have beendetermined to be suitable in practice of the invention. Preferably, inaccordance with practice of the invention, such genes are selected wherethere is a correlation between the level of gene expression and thenature and extent of a disease state or other undesired condition.

[0004] In accordance with one aspect of the invention, the clinicalspecimen is a sample of blood, tissue, or cerebrospinal fluid. Theclinical specimen may be a sample of blood, and derived therefrom, suchas plasma or serum sample or fraction.

[0005] In accordance with another aspect of the invention, theexpression levels of at least three preselected genes are measured. Inone embodiment, the expression level of at least one proinflammatorycytokine is measured. In another embodiment, the expression level of atleast three preselected proinflammatory cytokines is measured. In yetanother embodiment, the preselected proinflammatory cytokine genes areselected from the group consisting of TNF-α, IL-6, IL-1, IL-8, IP-10 andMIP-1α.

[0006] In accordance with another aspect of the invention, the clinicalcondition or syndrome is an inflammatory disorder. In one embodiment,the inflammatory disorder is a chronic inflammatory disorder. In anotherembodiment, the chronic inflammatory disorder is selected from the groupconsisting of chronic hepatitis, hepatitis B and C, chronic obstructivepulmonary disease, inflammatory mucosal disease, autoimmune disease,dementia, cardiovascular disease, and cancer. The inflammatory mucosaldisease may be selected from the group consisting of inflammatory boweldisease, Crohn's disease, and colitis. The dementia may be AIDS-relateddementia or Alzheimer's disease. The cancer may be selected from thegroup consisting of lymphoma, prostate cancer, and colon cancer. Inanother embodiment, the clinical condition is transplant rejection in apatient with an allograft. The allograft may be a heart, liver, kidney,or other organ.

[0007] In accordance with still another aspect of the invention, theclinical outcome that is determined is response to a therapeuticintervention. The therapeutic intervention may be treatment with a drug.The drug may be a stabilized chlorite solution. In particular, thestabilized chlorite solution may be WF-10.

[0008] In accordance with yet another aspect of the invention, theclinical condition or syndrome is HIV infection. In one embodiment, theclinical condition or syndrome is AIDS.

[0009] In accordance with a still further aspect of the invention, theindicated clinical outcome is the probability of patient survival at apredetermined date. In one embodiment, the indicated clinical outcome isthe probability of patient survival after six months.

[0010] In accordance with a further aspect of the invention, the levelsof gene expression are measured by a quantitative polymerase chainreaction. The polymerase chain reaction may be a reversetranscriptase/polymerase chain reaction. The polymerase chain reactionmay be carried out using fluorescent detection of the amplificationproducts. In one embodiment, the polymerase chain reaction may becarried out using a LightCycler® instrument, or using other appropriatetechnology.

[0011] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 shows a schematic of a macrophage activation cycle whereinmultiple steps occur during various forms of activation and recycling ofmacrophage function so as to achieve Balanced Macrophage Activation.

[0013]FIG. 2 shows the changes in gene expression of proinflammatorycytokines in a patient (#14) after treatment with WF10. The levels arereduced, indicating a good prognosis and good response to treatment.

[0014]FIG. 3 shows the changes in gene expression of proinflammatorycytokines in a patient (#15) after treatment with WF10. The levels arelow to begin with, and are unchanged with treatment, indicating thattherapy is unnecessary and that the patient has a good prognosis.

[0015]FIG. 4 summarizes characteristics of the patients studied inExample 2. Patients were being enrolled in a prospective Phase II studyevaluating the potential of WF10 for treatment of HCV disease. Baselineblood specimens were available from these patients with paired liverbiopsies for the study. All patient histories and specimens wereobtained in accordance with standard Committee on Human Researchapproved protocols. The acutely infected patient was not being evaluatedfor treatment by WF10 but presented to one of the study's referringphysicians. That patient's specimens were evaluated with the same humansubjects approval criteria. The 8 patients with chronic HCV infectionhad baseline demographic and laboratory data obtained. The patients werenot selected by any criteria except that those patients had blood drawnbefore enrollment in the WF10 clinical trial and had a liver biopsyperformed within 2 weeks of the blood draw. Laboratory values of HCVgene expression levels and liver function tests were also obtainedwithin this same 2-week window of time. Liver biopsies were obtainedfrom the patients evaluated by an independent pathologist, who scoredthe inflammation grade base on standard 4-point grading system. Only atthe end of the gene expression evaluation were all data regarding theliver biopsy, laboratory values, and gene expression values pooled forultimate data analysis. Only Patient 4 in this study had been treatedpreviously with interferon and Ribivarin and had not received anytreatment in the 3-month period prior to entry into the study.

[0016]FIG. 5 shows immune activation gene expression in PBMC frompatients with acute and chronic HCV infection.

[0017]FIG. 6A shows the actual baseline gene expression values and GESfor the patients whose data are summarized in FIG. 5.

[0018]FIG. 6B shows the actual induced gene expression values and GESfor the patients whose data are summarized in FIG. 7.

[0019]FIG. 7 shows induced gene expression in PBMCs obtained from acuteversus chronic HCV infected patients.

[0020]FIG. 8 shows the multigene expression score in unstimulated PBMCof patients with HCV disease.

[0021]FIG. 9 shows the scoring system used in MGES value calculations.

[0022]FIG. 10 shows multigene expression score in stimulated PBMC ofpatients with HCV disease.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides methods of determining clinicaloutcomes in patients by measuring levels of expression of a preselectedset of genes. The gene expression levels are compared to referencestandards and deviations from those standards are indicative of clinicaloutcomes. The gene expression levels can be measured in essentially anyclinical specimen, including tissue or fluid, such as cerebrospinalfluid. Surprisingly, however, the inventors have discovered that geneexpression levels measured in blood samples are indicative of clinicaloutcomes. This is surprising because the blood has typically beenconsidered to be a quiescent organ of the body, and that measurement ofgene expression levels in the blood has been thought to be an exerciseof little or no value. In particular, measurement of intracellular geneexpression levels in blood cells can be used. Of course, use of bloodmakes obtaining and analyzing clinical samples simple, convenient, andminimally invasive.

[0024] Measurement of gene expression levels

[0025] The gene expression levels used in the methods of the inventioncan be measured by any method now known or that is devised in the futurethat can provide quantitative information regarding the levels to bemeasured. The methods preferably are highly sensitive and providereproducible results. In one embodiment, methods based upon nucleic acidamplification technologies are used. In particular, methods based uponthe polymerase chain reaction (“PCR”) and related amplificationtechnologies, such as NASBA and other isothermal amplificationtechnologies, may be used. More particularly, so called “RT-PCR” methodsusing reverse transcription of mRNA followed by amplification of theresulting cDNA are contemplated.

[0026] Methods for carrying out quantitative PCR are known in the art.See, for example, U.S. Pat. Nos. 5,210,015 and 5,487,972 and EP 512334B1which are hereby incorporated by reference in their entirety. Commercialinstruments for carrying out quantitative PCR and RT-PCR are availablefrom PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City,Calif. 94404, from Roche Molecular Systems, Inc., 1145 Atlantic Avenue,Alameda, Calif. 94501, and from Roche Molecular Biochemicals,Indianapolis, Ind. In a particular embodiment, the LightCyclerinstrument from Roche Molecular Biochemicals is used. This instrumentcan be used following the manufacturer's instructions as describedbelow. Primer sets for amplification of any known gene can be designedusing methods that are well known in the art, for example, using genesequences from public databases such as GENBANK and using primer designsoftware such as OLIGO. Primer sets for many genes also are commerciallyavailable, for example from PE Applied Biosystems, Roche MolecularBiochemicals, Roche Diagnostics, and Search-LC (Heidelberg, Germany).

[0027] Samples for measuring gene expression levels

[0028] Any conveniently available tissue sample from a patient can beused for measurement of gene expression levels. In particularembodiments, the sample can be blood, cerebrospinal fluid, and cellulartissue derived from biopsy or from exfoliation such as from the cheekwall. In one embodiment, the sample is peripheral blood mononuclearcells, which are readily and easily available via minimally invasivemethods. Methods for preparing the sample for gene expression analysisare well known in the art, and can be carried out using commerciallyavailable kits.

[0029] Determining clinical outcomes

[0030] The gene expression levels obtained preferably are compared andnormalized against reference genes in the same sample. Typically,“housekeeping” genes such as actin, are used for this normalization.Other “housekeeping” genes are well known in the art, such as HPRT, CPB,and G6PD.

[0031] For determining clinical outcomes, the gene expression levelsobtained from the clinical sample (from the “test patient”) are comparedto levels in reference samples. The reference samples typically areobtained from healthy individuals who are disease free, or who are notsuffering from the same pathological condition or syndrome as the testpatient. Preferably, expression levels of the genes of interest aredetermined from a number of healthy individuals, and an average or meanis obtained. In a particular embodiment, the reference levels may bedetermined from individuals of the same sex and age as the test patient.In another embodiment, the reference levels may be obtained fromtabulated data, where those data are compiled from healthy patients ofappropriate sex and age.

[0032] The relative levels of gene expression that can be predictive (atype of indication) of clinical outcome can be higher or lower levels ofexpression. For example, it is shown below that increased levels ofexpression of proinflammatory cytokines, such as TNF-α, IL-6, IL-1,IL-8, IP-10 and MIP-1α are reflective of a poor clinical outcome (forexample, reduced expectation for long-term survival) for patientsinfected with HIV. When the patient is treated with an anti-HIVcompound, the relative levels of the proinflammatory cytokines can bemeasured again. If the levels of the cytokines is reduced, thisindicates that the patient is responding well to the treatment. In thiscase, the clinical outcome may be that the patient can cease anti-HIVtherapy, or reduce the dose of the anti-HIV compound. A lack of responsecan indicate that the patient will not respond to therapy, and thereforehas a poor prognosis, or that the dose of anti-HIV compound must beincreased, or additional therapeutic interventions must be used. A lackof response also may indicate that the progression of the disease hasnot been halted or slowed by therapy.

[0033] Although the present invention specifically contemplates that thelevels of proinflammatory cytokine expression can be measured and usedto predict clinical outcome, the skilled artisan will recognize that theinvention is not so limited. Thus, methods for identifying changes ingene expression are well known in the art, as described supra. Eventhough these methods are not sufficiently quantitative for use in thepresent invention, they can be used to predict those genes whoseexpression is changed in a disease state. Quantitative measurements,such as quantitative RT-PCR can then be used to measure the changes ingene expression. Those changes can be tracked in patients and correlatedwith clinical outcomes by methods that are well known in the art.

[0034] Thus, for any given disease, a predictive method for determiningclinical outcome can be developed. The discussion below describes thegene expression levels in macrophage activation, and describes how thoselevels, and their change upon treatment with a particular drug (WF10)provide information regarding clinical outcomes in patients sufferingfrom chronic inflammatory disease, such as chronic hepatitis, hepatitisB and C, chronic obstructive pulmonary disease, inflammatory mucosaldisease, autoimmune disease, dementia, cardiovascular disease, andcancer. The inflammatory mucosal disease may be selected from, forexample, inflammatory bowel disease, Crohn's disease, and colitis. Thedementia may be, for example, AIDS-related dementia or Alzheimer'sdisease. The cancer may be,, for example, lymphoma, prostate cancer, orcolon cancer. Example 2 below also demonstrates that the methods of theinvention can be used to predict clinical outcome in patients sufferingfrom hepatitis C. The methods of the invention may also be used todetect or predict clinical outcome of transplant rejection in patientsreceiving allografts, such as heart, liver, kidney, or other organs.Thus, measurement of altered, particularly increased, inflammatorycytokine gene expression is indicative of rejection of the allograft.Particular gene expression levels that can be measured to detectallograft rejection include IP10, TNFα, δ-IFN, and other macrophageinflammation genes.

[0035] Activation of macrophages

[0036] WF10 is a stabilized chlorite matrix approved for clinical use ina systemic form (WF10) and in a more dilute topical form (Oxoferrin) SeeKühne, Die erwünschte Sauerstoffaktivierung, dokumentiert am Beispielder Wundheilung: der Weg zur Oxoferin-Therapie. In: Elsmer E. F., BorsW., Wilmanns W. (eds.): Reaktive Sauerstoffspezies in der Medizin.Springer Verlag, Berlin 1986, pp. 5-15. WF10 has been approved inThailand for systemic administration to patients with post-radiationsyndrome and for supportive care in patients being treated for cancer.These indications have been studied extensively and reviewed. SeeRaffanti et al., Infection 26:201-206 (1998). Oxoferrin is usedtopically to enhance healing of chronic wounds such as diabetic ulcers.Hinz et al., Lancet 1: 825-828 (1986). The major target cells in thebody for WF10 reactivity are the macrophage and dendriticcellpopulations. They will be referred to simply as macrophages as both cellpopulations are derived from common precursor cells. The in vitro andclinical effects of WF10 are best understood in the context of the newlyproposed balanced-macrophage activation theory. FIG. 1 shows a schematicof a macrophage activation cycle wherein multiple steps occur duringvarious forms of activation and recycling of macrophage function so asto achieve Balanced Macrophage Activation. Each step in the macrophageactivation cycle is numbered (1-5) and is described sequentially.

[0037] 1. The first thing that occurs in a macrophage activation programis phagocytosis of foreign material. Macrophages engulf pathogenicorganisms such as bacteria, fungi and viruses. This is one of the oldestand most important functions of macrophages and is how the macrophagederived its name. “Macro” meaning big, and “phage” meaning eater,thereby conferring on the macrophage the term “Big Eater.” Uponsuccessful phagocytosis of a foreign substance, the macrophage processesthis material through a proteolytic pathway, cutting individual proteinsinto small peptides that then are involved in the second step ofmacrophage activation.

[0038] 2. Antigen presentation: After foreign materials have been cutinto peptides, macrophages present antigen to T lymphocytes utilizingthe major histocompatibility antigens class 1 (HLA) and class 2 (DR) andinitiate expansion of a normal immune response. T cell activationpredominantly occurs through this antigen-presenting-cell function.Standard cytotoxic T cells specific for virus infected cells, cancers orfungi are developed that ultimately lead to successful immunologicclearance of those foreign processes. This is represented in FIG. 1 asan active immune response. Upon successful activation of an activeimmune response, T cells express various activation antigens such asCD38 and secrete factors such as interleukin-2 (IL-2). IL2 allows Tcells to proliferate and gamma-interferon (γ-IFN) to cause furthermacrophage activation and step 3.

[0039] 3. Classical macrophage activation: A product of T cellactivation, gamma-interferon induces full inflammatory changes andclassical macrophage activation. This activation causes upregulation ofinflammatory cytokines such as IL1, IL6, and tumor necrosis factor(TNF). The macrophage in this state is extremely inflammatory and causessecondary effects such as fevers, and when chronically stimulated,weight loss and further non-specific activation of immunologicresponses.

[0040] 4. TH1 to TH2 (Active to Inhibitor T) shift: During theinitiation of a cellular response which ultimately leads to productionof cytotoxic T cells and T cells producing IL2 (TH1 cell), a secondmajor class of T cell, the TH2 cell, is induced which is involved inboth B cell activation as well as providing signals for the balancedmacrophage activation. Cytokines produced by the TH2 cells include IL4,IL5, IL6, and IL10. These factors cause B cell activation, B cellproliferation, hypergammaglobulinemia, up-regulation of IgE and allergicreactions and eosinophilia. A net result of excess IL10 production isshutting off of step 2 in the response shown in FIG. 1. The TH1 and TH2cell activation process occur virtually simultaneously in vitro (andlikely in vivo), however classical immunologic responsiveness asmeasured by T cell proliferation in vitro predominantly measures theTH1-like response. The TH2 response has as a key feature, the productionof IL4, which is known to activate the alternative macrophage activationpathway (AMAP). (Step 5)

[0041] 5. AMAP: The Alternative Macrophage Activation Pathway (reviewedin ref. 4) has the following features:

[0042] a. the production of angiogenic factors

[0043] b. inhibition of T cell responses

[0044] c. associated down-regulation of inflammatory-mediator productioncharacteristic of classically-activated macrophages described in step 3.

[0045] The Alternative Macrophage Activation Pathway recently has beenconfirmed as a distinct pathway with the cloning and molecular studiesof AMAC-1 (Alternative Macrophage Activation Chemokine-1) (Kodelja etal, Journal of Immunology 160:1411-1418 (1998)), also known asmacrophage inflammatory protein 4 (MIP-4). Only in macrophages inducedto undergo Alternative Macrophage Activation Pathway has the geneAMAC-1/MIP-4 been detected. A secondary byproduct of AlternativeMacrophage Activation Pathway is the appearance of phagocytosis inmacrophages that have been induced to undergo Alternative MacrophageActivation Pathway induction. This byproduct potentially signals thecomplete recycling of the Balanced Macrophage Activation pathway.

[0046] This scheme, wherein macrophage involvement in immunologicresponses goes from steps 1 through 5, is proposed as a cycle in thebalanced macrophage activation theory. Because of its cyclic nature,normal immunologic processes do not overemphasize any particular step inthe pathway but remain generally in balance.

[0047] Diseases involving Balanced Macrophage Activation disruption

[0048] Balanced Macrophage Activation is disrupted by a variety ofpathologic processes and Balanced Macrophage Activation imbalance isresponsible for many manifestations of chronic disease. Examples ofthese imbalances are as follows:

[0049] Chronic viral infections: Steps 2 and 3 in FIG. 1 are continuallystimulated when a foreign virus cannot be cleared by a successful immuneresponse that would re-establish Balanced Macrophage Activation. Thisimmunologic overstimulation would predictably lead to pathologicsequelae such as cirrhosis and hepatoma in chronic hepatitis B & Cinfections and profound immune dysregulation in HIV disease. After longperiods of time wherein steps 2 and 3 are overemphasized, there would bea predicted shortage of cells to accomplish steps 5 and 1. There alsowould be an initial overdrive of the TH1 cell population with theappearance of highly activated T cells. An overactivation of step 3would clinically appear as chronic fever with associated weight loss.Patients with chronic viral infections such as those with HIV also havebeen observed to have a dramatic TH1 to TH2 shift as described in step 4for FIG. 1. The Balanced Macrophage Activation theory predicts that thisshift is compensatory in nature with the T cells attempting to regulateBalanced Macrophage Activation through production of IL4, the cytokinethat normally induces step 5. The conditions suffered by patients withchronic viral diseases would then be byproducts of a chronicinflammatory state. These would include overproduction of inflammatorymediators and inflammatory cytokines that cause secondaryimmunopathogenic changes. Such changes are observed in HIV disease whereexcessive inflammatory states drive development of dementia, kidneydisease, lymphoma, and wasting syndromes that are secondary tohyperactivity of the macrophage inflammation compartment. A secondarybyproduct of chronic viral disease would be the exhaustion of cells insteps 5 and 1 as noted above. This result would decrease the rate ofwound healing and decrease associated angiogenesis and phagocytosis.Fewer cells capable of phagocytosing material would allow new infectiousorganisms such as bacteria and fungus to be poorly cleared byindividuals with chronic viral diseases.

[0050] Autoimmune disease: Autoimmune diseases are similar to chronicviral diseases in that there is an overstimulation of immunoreactivelymphocytes with associated inflammation. Autoimmune disease has formany years been thought of as a chronic viral-like disease, however novirus has to date been isolated as an initiator of these types ofdiseases. These diseases include systemic lupus (SLE), post-radiationsyndrome, and a variety of autoimmune kidney diseases, etc. Features ofsome autoimmune diseases are the presence of hypergammaglobulinemia,elevated IgE and eosinophilia as described above in FIG. 1 step 4. Thisresult may occur as the by-products of a compensatory TH1 to TH2 shiftwhen the body attempts to reestablish Balanced Macrophage Activation.

[0051] Allergic reactions: The most serious allergic reaction is asthmawherein overstimulation of step 2 with environmental antigens in thelung leads to inappropriate local macrophage inflammatory changes and Tcell activation in lung tissues. These lung tissues are harmed byinflammatory mediators produced in step 3. Normally lung macrophagesconstitutively have the Alternative Macrophage Activation Pathwayinduced) (as shown in step 5) and they therefore are less susceptible tosteps 2 and 3 as shown in FIG. 1. However, in patients with allergiesthese reactions (steps 2& 3) are allowed to occur. Asthmatic patientsalso have a TH1 to TH2 shift with associated eosinophilia. This reactionis predicted by the Balanced Macrophage Activation theory to becompensatory when it attempts to shift lung macrophages from steps 2 & 3through 4 into step 5.

[0052] Immune deficiency associated bacterial and fungal infections: Ifsteps 2 and 3 from FIG. 1 are increased, over time there will be fewercells in steps 5 and 1 capable of phagocytosis and reinitiation ofimmune responses. The decreased number of cells capable of phagocytosingbacteria and fungus makes patient survival in the presence ofimmunodeficiency quite problematic. Antibiotic therapy directed againstbacteria and fungus works inefficiently in vivo unless the invadingorganisms have been phagocytosed by macrophages or granulocytes. Themost commonly used antifungal drug, amphotericin B, does not work at allunless fungus has been engulfed by a phagocytic cell. Patients withadvanced HIV disease are susceptible to invasive fungal infectionsmostly because of inefficient phagocytosis. A parallel disease processis induced in patients with organ transplants who receive cyclosporin Aand Prednisone for treatment of graft rejection. These patients areimmunosuppressed and if they develop invasive bacterial or fungalinfections will have their macrophages shifted toward steps 2 and 3 andsimilarly cannot recycle and achieve Balanced Macrophage Activation,which would allow phagocytosis and reinitiation of immunologicresponsiveness.

[0053] Chronic wounds: The best example of this class of disease isobserved in patients with diabetes or those who are bedridden. Chronicdiabetic and pressure ulcers develop and macrophages within those woundsexhibit changes consistent with step 3 in FIG. 1. Goerdt et al.,Immunity 10: 137-142 (1999). Wounds will not heal if step 3 cannot beshifted through step 5 wherein angiogenic factors are produced to allowblood vessel growth and healing. Similarly if macrophages within achronic wound have been shifted from step 1 to 3, phagocytic cells willnot be present to allow clearance of dead and dying material withinwounds so as to speed the healing process.

[0054] Cancer: A variety of cancers are outgrowths of chronicinflammation. Examples include lymphoma, which represents outgrowths ofantigenoverdriven lymphocytes, and prostate cancer, which evolves fromchronic prostatitis. In both cases steps 2 & 3 provide chronic growthstimuli.

[0055] In vitro studies of WF10 activity on immune function:

[0056] WF10 completely blocked antigen activation of T cellresponsiveness at levels easily achievable in vivo. McGrath et al.,Transplantation Proceedings, 30: 4200-4204.(1998). This inhibition of Tcell activation only occurred when T cells and macrophages were placedtogether with the foreign antigen, and occurred instantly or even whenadded at day 6 of a 7-day T cell activation assay. These data suggestthat WF10 is extremely potent at inhibiting processes fundamental tonormal T cell activation as shown in step 2 for FIG. 1.

[0057] WF10 caused downregulation of inflammatory cytokine production byinflammatory macrophages as described in step 3 for FIG. 1. McGrath etal. Abstract #2046, Keystone Symposia on Molecular and Cellular Biology,Park City, Utah, Mar. 13-19, 1998. Studies are currently underway totest whether WF10 causes upregulation of AMAC-1, thereby converting step3 to 5 and completion of the BMA cycle in vitro.

[0058] The use of WF10 to achieve BMA in vivo:

[0059] WF10 and Oxoferrin have been used extensively for many years totreat chronic disease in humans. Oxoferrin was approved for topical usein chronic wounds in the late 1980's. To date Oxoferrin has beensuccessful in inducing rapid healing of chronic wounds includingdiabetic and pressure ulcers. Oxoferrin is thought to work throughachieving Balanced Macrophage Activation with associated upregulation ofangiogenic factors and macrophage phagocytosis. WF10 was approved inThailand for systemic use in 1997 for treatment of post-radiationsyndrome (PRS). Post-radiation syndrome occurs as a late complication inorgans that have received X-ray therapy. Up to 15% of patients who havehad lower abdominal irradiation for cervical or prostate cancer developPRS associated bleeding from the bladder and rectum 6 months to 10 yearsafter that radiation. Histologic analysis shows that the bleeding iscaused by a local autoimmune process within the small arteries(endarteritis). This leads to death of tissues within the radiationfield and causes bleeding, for example, into the bladder or rectum.Patients sometimes respond temporarily to steroids. However, in studiesperformed in Thailand there was nearly a 100% complete response rate tosystemic administration of WF10 in women with hemorrhagic cystitissecondary to post-radiation syndrome. Unlike steroid treatment, which isassociated with some symptomatic relief, WF10 administration to date hasbeen curative for patients with post-radiation syndrome.

[0060] Advanced clinical studies of WF10 currently are underway in theUnited States for treatment of patients with HIV disease. Patients whoreceived two cycles of WF10 showed chronic immunologic changesconsistent with induction of Balanced Macrophage Activation. Herndier etal, Abstract #22417, 12^(th) World AIDS Conference, Geneva, Jun. 28-Jul.3, 1998. Patients with HIV disease, a chronic viral infection, typicallyshow inappropriate elevated T cell activation and decreased rates ofmacrophage phagocytosis in end-stage disease. In Phase II studiesconducted at San Francisco General Hospital, WF10 administration wasassociated with dramatic down-regulation of all inappropriately elevatedimmunologic activation markers with up-regulation of macrophagephagocytosis in patients who had low baseline levels of phagocytosis atthe initiation of study. These results were consistent with induction ofBalanced Macrophage Activation and will lead to further in vitro studiesto test components of the Balanced Macrophage Activation theory.

[0061] In conclusion, it appears that diseases affecting BalancedMacrophage Activation are common and that many of the side effects ofchronic disease occur because of toxic side effects mediated bymacrophages accumulating in any one of the steps described above.Currently, WF10 is the only new drug in clinical development that mayaffect Balanced Macrophage Activation and might be expected to changethe clinical outcome of the diseases in sections A through F describedabove.

[0062] The use of an aqueous solution containing a stabilized chloritesolution for treating wounds and infections is known in the art. U.S.Pat. Nos. 4,507,285 and 4,725,437, the disclosures of which areincorporated by reference herein in their entirety, and EP 0 200 157,the disclosure of which also is incorporated by reference herein in itsentirety, describe the use of a stabilized chlorite solution instimulating the wound healing response in humans, as well as in treatinginfections caused by parasites, fungi, bacteria, viruses and/ormycoplasma. Kühne et al., European Patent No. 200,156, the disclosure ofwhich is incorporated by reference herein in its entirety, describes theuse of a stabilized chlorite solution in conjunction with radiationtherapy to aid in repairing damaged irradiated tissue and reducing sideeffects.

[0063] A preferred embodiment of the treatment of this invention entailsadministration to a mammal in need thereof, an aqueous solution of aproduct that has been termed “tetrachlorodecaoxygen anion complex,”commonly abbreviated as “TCDO.” This substance can be prepared using theprocedures described in Example 1 of U.S. Pat. No. 4,507,285 (“the '285patent”), and is a water clear liquid, miscible with alcohols, and has amelting point of −3° C. The Raman spectrum shows bands of 403, 802(chlorite) and 1562 cm/⁻¹ (activated oxygen). The skilled artisan willrecognize that any chemically stabilized chlorite solution can be usedin the methods of the present invention, and that the scope of theinvention is not limited to use of the product described in the '285patent.

[0064] The present invention, thus generally described, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention. In the examples, “WF10” denotes an aqueousstabilized chlorite solution.

EXAMPLE 1 METHODS

[0065] Sample preparation

[0066] Mononuclear cells (MNC) were isolated on a Histopaque™1077density gradient using Leuco Sep tubes (#227290, Greiner Labortechnik,Frikenhausen, Germany) and 2×10⁶ cells were resuspended in RPMI 1640with 10% FCS. Cultures were stimulated either with a mitogenic anti-CD2mixture (AICD2M1, AICD2M2 final concentration 1 μg/ml each and 11F1 300ng/ml), anti-CD3 (OKT-3, 100 ng/ml) or 10 ng/ml PMA and 0.5 μg/mlionomycin. Unstimulated and stimulated cultures were incubated in thepresence or absence of WF10 at a final concentration of 1:300 for 3 hrsat 37° C. in 7% CO₂. Cells were harvested, resuspended in 200 μl PBS and400 μl of High-Pure lysis solution was added. Resulting lysates werestored at −70° C. After thawing at 37° C. for 10 minutes, RNA wasextracted using a total RNA isolation kit and RNA was eluted from thespin column in a volume of 50 μl. An aliquot of 8.2 μl RNA was reversetranscribed using AMV-RT and oligo-(dT) as primer (cDNA synthesis kit).As a control, a reaction was performed without reverse transcriptase(no-RT control). After termination of cDNA synthesis the reaction mixwas diluted to a final volume of 500 μl and stored at −20° C. until PCRanalysis.

[0067] LightCyder® PCR

[0068] Target sequences were amplified using LightCycler® Primer Sets(Search-LC, Heidelberg, Germany) with the LightCycler FastStart DNA SybrGreen 1 Kit (Roche Diagnostics, Indianapolis, Ind.) according to themanufacturer's protocol. Input was normalized by the average expressionof the four housekeeping genes β-actin, HPRT, G6PDH and Cyclophilin B.

[0069] Overview of Study Design

[0070] Eighteen HIV-infected adults with CD4+ counts greater than 50cells/mm³ treated with approved anti-HIV medications are enrolled in anopenlabel, single center study. Participants are involved in the primaryphase of the study for approximately 12 weeks. Patients are stratifiedinto two cohorts of nine patients: nine patients have CD4+ counts >300cells/mm³ and nine patients have CD4+ counts >50 and <300 cells/mm³.Only patients with a viral load of <20,000 copies/mL are enrolled in thestudy. Enrollment is limited to nine patients with plasma HIV RNA belowthe limit of assay delectability.

[0071] After screening evaluations are completed, eligible patientsattend study visits on Days 1, 2, and 4. From Days 8 through 12,patients receive one cycle of WF10 0.5 mL per kg/bw diluted into 250-500mL normal saline administered by intravenous infusion. Patients thenattend study visits on Days 15, 17, 19, 22, 24, and 26.

[0072] From Days 29 through 33, patients receive a second cycle of WF100.5 mL per kg/bw diluted into 250-500 mL normal saline administered byintravenous infusion. Patients then attend study visits on Days 36, 38,40, and 47 (final visit). Patients have a 48-hour window in which toreturn for the final visit on Day 47.

[0073] Immune function, measured on days 1, 8, 11, 15, 22, 29, 31, 40and 47, is defined as the measurement of phagocytic index usingfluorescein-labeled E. coli, T cell activation with phytohemagglutinin,lymphocyte immune phenotyping (detecting CD3, CD4, CD8, CD14, CD20,CD28, CD38, CD56, CD69), DR, TNF and monocyte quantitation.

[0074] The health status of all patients is followed up by monthlytelephone calls for one year after Day 47.

Results

[0075] Clinical Trial

[0076] Of the patients who completed the 47 day study, seven had initialCD4 counts <300/μl, and those patients showed the most dramaticimmunologic parameter response to two 5 day cycles of i.v. WF10. CD4 andCD8 counts increased significantly and the CD8 cell increase wascompletely reflected by an increase in the CD+E, 8/38 negative subset.There was an overall dramatic in median CD38 expression with noassociated change in HIV viral load during the course of this trial.Other measures of immunologic activation were similarly decreased withan observed significant drop in CD14/DR, CD20/DR and CD4/38 mean levelsof cell associated fluorescence.

[0077] Gene Expression Analysis of WF10 Trial PBMC

[0078] PBMC associated inflammatory gene expression was evaluated onfrozen and subsequently extracted cell preparations using Lightcyclerbased technology. FIG. 2 shows relative levels of a series ofproinflammatory genes(to internal housekeeping genes, actin, G6PD, CPB,HPRT) in a patient(#14) who had a 50% decrease in the CD8/38+ cellsubset compared to a patient(#15) who had no change of CD8/38 during the47 days of the trial. The gene expression levels in patient 15 are shownin FIG. 3. There was an overall highly significant correlation betweendecrease in CD8/38 level and the change in expression of macrophageproinflammatory genes such as those shown in FIG. 2.

[0079] In Vitro Effects of WF10 on PBMC and CD14 Cell Gene Expression

[0080] WF10 was tested in vitro on PBMC's exposed to anti-CD2, anti-CD3and PMA/ionomycin to determine effects of the drug on T cell activation.WF10 was used at a final concentration of 1:300, a dose easily achievedduring the clinical trial and PBMC's were harvested three hours andaffinity purified CD14 cells 18 hours later for RNA extraction andRT-PCR. WF10 effects on 11 normal blood donor PBMC's were expressed asLC-Index which represents up to a 5 fold change from baseline un(WF10 )treated but stimulated specimens. A consistent down regulation ofinduced lymphostimulatory cytokines IL-2 and IL-17 was observed, with aconsistent pattern of IL-1β, IL-8, MIP-1a and thioredoxin (TRX)upregulation. Because the upregulations appeared to be macrophageinflammatory mediators, purified CD14 cells were exposed to 1:300 WF10for 18 hours and evaluated for increased gene expression. Three of theseven CD14 preparations had an approximate decrease in the 4housekeeping gene levels of 90%. Three CD14 specimens also had dramaticupregulation of the 4 apoptosis genes evaluated and parallel PI uptakestudies confirmed CD14 cell apoptosis occurring in those cultures. Thegene expression levels of PBMC's used for the macrophage studies beforethey were treated with WF10 also were determined. It was observed thatthose specimens that became apoptotic had significantly higher levels ofpretreatment inflammatory gene expression than those that did notundergo apoptosis.

[0081] WF10 Treatment In Vitro Leads to AMAC-1 Upregulation

[0082] Apoptotic cells are the most potent stimulus for macrophages tounder alternative (anti-inflammatory) activation and phagocytosis.Alternative activation has been associated with induction by the Th2cytokine IL-4 and causes a complete block in inflammatory geneexpression and antigen induced T cell activaton. Purified macrophageswere exposed to a 1:200 dilution of WF10 and AMAC-1 (specific for thealternative pathway, AMAP) gene expressioin was assessed up to 21 dayslater. WF10 dramatically augmented the AMAC-1 expression induced bymacrophage treatment with IL-4.

[0083] These data suggest that WF10 administration changes expression ofmacrophage proinflammatory gene expression in a pattern that parallelschanges in CD8/38 levels in vivo. In vitro, WF10 caused dramatic changesin a wide variety of immunologically active genes leading to apoptosisin CD14 cells in a subset of preactivated patient specimens andconsistent down regulation of lymphostimulatory genes in PBMCs. It isapparent that WF10 regulates inappropriate inflammation associated withchronic inflammatory diseases such as HIV disease through overactivationinduced CD14 cell death with compensatory induction of thealternative(anti-inflammatory) pathway of macrophage activation.

[0084] In conclusion, the studies described above demonstrate that

[0085] 1) WF10 administration to HIV+ patients (<300 CD4 cells/μl) wasassociated with significant:

[0086] a) Increase in CD4, CD8, CD8/38-cell numbers

[0087] b) Decrease in CD20/DR, CD14/DR, CD4/38 mean fluorescence/cell

[0088] c) Decreased PHA activation

[0089] 2) Lightcycler system RT-PCR of PBMC's showed drug-associateddown regulation of macrophage inflammatory gene expression (TNF-α,etc.).

[0090] 3) In vitro studies of WF10 effects on normal PBMC (3h) and CD14(18h) cells showed:

[0091] a) Activated PBMC's: Down regulation of lymphostimulatorycytokines IL-2 and IL-17. Upregulation of macrophage inflammatory genesas well as the anti-apoptotic (antioxidant) gene thioredoxin (TRX)

[0092] b) Cultured CD14 cells: Upregulation of apoptotic genes (BAX,BCL-X1, CD95, CD95L) in specimens containing elevated pre-treatmentlevels of inflammatory genes with associated apoptotic cell (CD14)death.

[0093] 4) WF10 caused late upregulation of the alternative macrophageactivation gene AMAC-1 in isolated CD14 cells.

[0094] 5) Without being bound by any theory, the inventors believe thatWF10 induction of macrophage cell death in specimens containing elevatedinflammatory gene expression leads to compensatory AMAP induction inmacrophages in response to the acute apoptosis of inflammatorymacrophages. Accordingly, WF10 causes downregulation of inflammationthrough acute upregulation of inflammatory gene expression, cell deaththrough apoptosis, downregulation of lymphostimulatory genes andcompensatory macrophage differentiation change to the AMAP,anti-inflammatory pathway.

[0095] These observations show that WF10 caused reversal of pathologicinflammation (associated with HIV disease progression) in vivo whichover time would be expected to show a long term clinical benefit. Thechange in immunologic state was documented through use of thequantitative gene expression technique. This test would be utilized inthe long term care of treated patients to rapidly identify the return ofa pathologic state requiring further WF10 therapy.

EXAMPLE 2 Summary

[0096] Peripheral blood mononuclear cells were obtained from the 9patients described in FIG. 4, as well as from 20 normal blood donors.Gene expression was assessed for baseline as well as PMA/ionomycinstimulated cells as described above. Quantitative evaluation was basedon use of 2 housekeeping genes (β-Actin, CPB) to serve as controls foroverall cellular gene expression. “Test” gene expression wassubsequently normalized to a standard “housekeeping gene” control level.Thirteen genes associated with macrophage and T cell activation wereselected for evaluation in this study because of their function inprimary immunologic responses and chronic inflammation. The genes wereIL-1, IL-2, IL-4, IL-8, IL-10, IP10, MCSF, TNFα, γ-IFN, MIP-1α, MIP-2α,MRP-14, and TGF-β. Normal values for housekeeping genes as well asinflammatory genes were established by evaluating gene expressionpatterns from 20 normal blood donors (see FIG. 5).

[0097] Baseline gene expression was assessed in the patient with acuteHCV infection. The quantitative analysis is shown in FIG. 5 as a geneexpression score (GES) wherein the quantitative value obtained for eachgene was given a score based on its ratio to the mean normal expressedgene level determined in the 20 normal blood donors. The highest levelof gene expression observed in the acutely infected patient was theIP-10 gene, known to be induced specifically by γ-interferon. Associatedwith this elevation of IP-10 was the expected elevation of γ-interferonas well as elevation of the majority of macrophage inflammatory genesassessed (for example, GM-CSF and TNF-α). Genes expressed atbaseline/normal values included IL-2, IL-4, IL-8, and IL10. Notably,this gene expression pattern was observed in unstimulated cells isolatedfrom patient blood and not liver or lymph node.

[0098] The average gene expression values from blood of the 8chronically infected patients showed a pattern of gene expressionsimilar to the acute infection specimen consistent with an ongoingimmunologic response. The actual GES values varied widely from patientto patient with chronic HCV infection, however. The average values foreach gene are shown in FIG. 5. The actual gene expression values and GESare shown in FIG. 6A (baseline) and FIG. 6B (PMA induced). This ongoingresponse detected in the blood, if present within the liver, could leadto progressive inflammatory damage consistent with that observed inprogressive HCV liver disease.

[0099] Earlier studies using liver biopsy analysis predicted that T cellhyperactivation would be observed in patients with HCV disease. SeeBurgio et al., Hepatology 27:1600-6 (1998); McGuinness et al. Gut46:260-9 (2000). To test this prediction, peripheral blood monouclearcells from acute and chronic HCV disease patients was stimulated withPMA and ionomycin. The gene expression pattern observed in the patientwith acute HCV infection (shown in FIG. 7) was unexpected. Thus the IL-2and IL-10 gene expression induced in the acutely infected patient wasdramatically lower than control values (outside of normal range of GES)suggesting a substantial inhibition of T cell activation associated withacute HCV infection. This inhibition of T cell activation and inhibitedγ-interferon production was coupled with persistent elevation inmacrophage inflammatory cytokine as well as chemokine genes, such asIP-10, that normally are induced by γ-interferon. This observationsuggests that the IP-10 values observed in FIG. 5 along with anelevation of γ-interferon gene expression may not have been the productof T cells as the inducibility of this gene is dramatically inhibited.The only other source of interferon gene expression in blood cells otherthan T cells would theoretically be the natural killer cell populationthat also is known to be active in initiating immune responses invirally infected patients. Like the inhibition of T cell activationobserved in acute HCV infection, the average GES of the 8 patients withchronic HCV infection showed a pattern of inhibited T cell activationand chronic macrophage activation.

[0100] The data shown in FIGS. 6B and 7 contrast with prior reportedexperiments in patients with HCV liver disease that showed elevation ofIL-2 levels and hyperactivity of T cells at the protein level. SeeMartin et al., Cytokine 11:267-73 (1999). To determine whether theobserved gene expression patterns would correlate with degree of liverinflammation pathology, the corresponding liver biopsies were read andgiven inflammatory grade scores using standard assessment criteria. FIG.8 shows the correlation between liver inflammation score and a scoringsystem based on utilizing GES values from inflammatory genes asdetermined in the current study. (FIG. 9) A high degree of correlationwas found between the MGES (multiple gene expression score) and thedegree of liver inflammation.

[0101] The degree of histologic liver inflammation was shown to beassociated with the induced gene expression analysis. (FIG. 10) In earlystage I disease, the strongest inhibition of induced gene expression wasobserved. During progression of liver inflammation that degree ofinhibition gradually reverses, and by stage III inflammation the degreeof gene activation was elevated, consistent with prior reports.Induction of IL-10 expression was persistently low even in late stagepatients (FIG. 6B).

[0102] To test the relationship between the MGES evaluations and thecurrent standard of care measurements (i.e., the ALT blood measurementand HCV quantitative viral load measurement), the ALT values and HCVviral loads for each of the patients shown in FIG. 4 were correlatedwith degree of liver inflammation similar to the studies shown in FIGS.8 and 10. Neither the ALT measurement (r²:24) nor the viral loadquantitation (r²:0.15) showed correlations with liver biopsyinflammation score, in marked contrast to MGES (r²:0.94 and 0.78 forFIGS. 8 and 10 respectively).

[0103] These data demonstrate a new testing system for evaluation ofpatients with HCV disease. The high degree of correlation of blood basedgene expression patterns with liver inflammation allows staging ofpatients with HCV infection. In particular, the use of rapid RT-PCRmethods provides a clinical parameter for assessing the degree of liverinflammation in situ that is faster, less dangerous and less expensivethan prior methods.

[0104] Materials and methods

[0105] Immune activation gene expression in PBMC from patients withacute and chronic HCV infection.

[0106] Blood specimens were obtained from 1 patient with acute infectionand the 8 patients described in FIG. 4. After Ficoll Hypaque separationthe PBMCs were placed at 37° in RPMI 1640 with 10% fetal calf serum for3 hours prior to washing and RNA extraction. The same procedure wasperformed on blood obtained from 20 normal blood bank donors seen in theHeidelberg, Germany, University Blood Bank. cDNA was synthesized andLightCycler based PCR performed as described above. In each specimen, 4internal housekeeping genes were included in the RT-PCR analysis; theseincluded the β-actin, HPRT, CPB, and G6PD genes. These 4 genes wereutilized to standardize the amount of RNA contained in each specimen andall gene expression scores (GES) were determined utilizing RNA copynumbers based on the internal standardized housekeeping gene copy numbernormalized data. Based on the standardized copy number of housekeepinggenes, a calculated copy number was derived for each of the genes shownin FIG. 6.

[0107] Thirteen genes were evaluated utilizing PCR primers (Search-LC,Heidelberg, Germany). The mean gene expression value obtained for 20normal blood donors was established as a gene expression score of 1 foreach of the 13 genes evaluated. Data shown in FIG. 5 represent thededuced copy number of the acute (A) and mean of the 8 chronic (C) HCVinfected patient blood specimens. Below each value shown graphically inFIG. 5 is the GES range of the 20 normal blood donors from the absolutelowest to the highest of the 20 values. The data plotted in FIG. 5represent a single determination of gene expression from all patients,with 1 representing an acute infection (A) and 8 patients combinedrepresenting the mean value from chronically infected patients. The geneexpression values obtained from the chronically infected patients werewidely variable and values are shown in FIG. 6.

[0108] Induced gene expression in PBMCs obtained from acute versuschronic HCV infected patients.

[0109] Blood was obtained as described above from patients with acuteand chronic HCV infection as well as 20 normal blood donors. PBMCs wereisolated, and placed into culture at 37° with PMA and ionamycin aspreviously described to induce acute T cell activation. After 3 hours ofincubation the cells were harvested, RNA extracted, andLightCycler-based PCR performed as described above. The GES shown inFIG. 7 was calculated based on normalization of gene expression based onhousekeeping gene expression. The data shown in FIG. 7 represent a GESfor the acute and mean of the 8 patients with chronic HCV infectioncompared to the mean of the GES calculated from the 20 normal blooddonor specimens. Also shown below FIG. 7 is the range from the lowest tothe highest values for each of the genes evaluated for the 20 normalblood donor patients. The data shown in FIG. 7 are broken into 2categories: genes representing T cell activation as compared to genesrepresenting macrophage activation. The acute infection (A) is comparedin FIG. 10 to the mean value of the 8 patients with chronic HCVinfection. The values for the chronically infected patients were highlyvariable and are shown in FIG. 6B.

[0110] Multigene expression score in unstinulated PBMC of patients withHCV disease.

[0111] The gene expression values shown in FIG. 6 with the deduced GESvalues were stratified based on the liver inflammation score obtainedindependently from a pathologist uninvolved in the gene expressionevaluation. A multiple gene expression score (MGES) was determinedutilizing gene expression values from the following genes from thepatients shown in FIG. 4 (TNFα, IP10, MIP-2α, interferon-γ, and MRP14).The GES of each patient's expressed gene involved in the MGEScalculation is shown in FIG. 9. The MGES calculated scores for eachpatient were then plotted based on a liver inflammation score determinedby independent pathologic evaluation and this data was plotted and areshown in FIG. 8. The 20 normal patients were also given MGES scoresrepresented in the figure as normal range which showed MGES from 0 to 2with a mean of 1. Patient with acute HCV infection is shown with an MGESscore of 10. The statistical relationship between the MGES score andliver inflammation score is shown as an r² value.

[0112] Multigene expression score in unstimulated PBMC of patients withHCV disease.

[0113] Similar to the MGES procedure utilized for data shown in FIG. 8,an MGES was determined based on the induced GES scores as shown in FIG.9. MGES determinations were based on the calculated GES evaluated foreach individual gene being then converted into an MGES scorecombination, including all 3 genes. The normal range is shown anddesignated normal range with the mean of 1 with the range from −3 to 0.Acute HCV infection is shown with a calculated MGES of −2.5. Thestatistical relationship between the MGES score and liver inflammationscore is shown as an r² value.

[0114] While the invention has been described in detail with referenceto the examples and particularly preferred embodiments, those skilled inthe art will appreciate that various modifications can be made to theinvention without departing from the spirit and scope thereof. Alldocuments referred to above are incorporated by reference.

What is claimed is:
 1. A method of evaluating a clinical outcome for apatient suffering from a clinical condition or syndrome, comprising thesteps of (a) providing a clinical specimen from said patient; (b)measuring the levels of intracellular expression of a preselected set ofgenes in cells in said clinical specimen; and (c) comparing said levelsof expression against a set of reference expression levels, wherein adeviation of the level of expression of one or more of said preselectedset of genes is indicative of clinical outcome for said patient.
 2. Amethod according to claim 1 , wherein said clinical specimen is a sampleof blood, tissue, or cerebrospinal fluid.
 3. A method according to claim2 , wherein said clinical specimen is a sample of blood.
 4. A methodaccording to claim 1 , wherein the expression levels of at least threegenes are measured.
 5. A method according to claim 1 , wherein theexpression level of at least one proinflammatory cytokine is measured.6. A method according to claim 4 , wherein the expression level of atleast three proinflammatory cytokines is measured.
 7. A method accordingto claim 6 , wherein said genes are selected from the group consistingof TNF-α, IL-6, IL-1, IL-8, IP-10 and MIP-1 α.
 8. A method according toclaim 1 , wherein said clinical condition or syndrome is an inflammatorydisorder.
 9. A method according to claim 8 , wherein said inflammatorydisorder is a chronic inflammatory disorder.
 10. A method according toclaim 9 , wherein said chronic inflammatory disorder is selected fromthe group consisting of chronic hepatitis, hepatitis B and C, chronicobstructive pulmonary disease, inflammatory mucosal disease, autoimmunedisease, dementia, cardiovascular disease, and cancer.
 11. A methodaccording to claim 10 , wherein said inflammatory mucosal disease isselected from the group consisting of inflammatory bowel disease,Crohn's disease, and colitis.
 12. A method according to claim 10 ,wherein said dementia is AIDS-related dementia or Alzheimer's disease.13. A method according to claim 10 , wherein said cancer is selectedfrom the group consisting of lymphoma, prostate cancer, and coloncancer.
 14. A method according to claim 1 , wherein said clinicaloutcome is response to a therapeutic intervention.
 15. A methodaccording to claim 14 , wherein said therapeutic intervention istreatment with a drug.
 16. A method according to claim 15 , wherein saiddrug is a stabilized chlorite solution.
 17. A method according to claim16 , wherein said stabilized chlorite solution is WF-10.
 18. A methodaccording to claim 1 , wherein said clinical condition or syndrome isHIV infection.
 19. A method according to claim 18 , wherein saidclinical condition or syndrome is AIDS.
 20. A method according to claim1 , wherein said predicted clinical outcome is the probability ofpatient survival at a predetermined date.
 21. A method according toclaim 15 , wherein said predicted clinical outcome is the probability ofpatient survival after six months.
 22. A method according to claim 1wherein the levels of gene expression are measured by a quantitativepolymerase chain reaction.
 23. A method according to claim 22 , whereinthe polymerase chain reaction is a reverse transcriptase/polymerasechain reaction.
 24. A method according to claim 23 , wherein saidpolymerase chain reaction is carried out using fluorescent detection ofthe amplification products.
 25. A method according to claim 1 , whereinsaid clinical condition is allograft rejection.